This invention is based upon the discovery by the inventors of the embC, embA, and embB genes that comprise the embCAB operon, and novel proteins encoded by the embCAB operon which are associated with the biosynthesis of the mycobacterial cell wall and are involved in resistance to the antimycobacterial drug ethambutol (EMB). The discovery of the embCAB operon and the proteins encoded by the operon will have important implications in the diagnosis and treatment of drug-resistant mycobacterial strains.
EMB is a selective antimycobacterial drug recommended for clinical use in 1996 (Karlson, A. G., Am Rev Resp Dis 84, 905-906 (1961)). Today, EMB remains an important component of tuberculosis treatment programs. Unfortunately, resistance to ethambutol has been described in 2-4% of clinical isolates of M. tuberculosis in the USA and other countries, and is prevalent among isolates from patients with multidrug-resistant tuberculosis (Bloch, A B., Cauthen, G M., Onorato, I M., et al. Nationwide survey of drug-resistant tuberculosis in the United States. JAMA 271, 665-671 (1994)).
EMB targets the mycobacterial cell wall, a unique structure among prokaryotes which consists of an outer layer of mycolic acids covalently bound to peptidoglycan via the arabinogalactan (Besra, G. S. & Chatterjee, D. in Tuberculosis. Pathogenesis, protection, and control (ed Bloom, B. R.) 285-306 (ASM Press, Washington D.C., 1994)). Lipoarabinomannan, another cell wall component of significant biological importance, shares with arabinogalactan the overall structure of the arabinan polymer (Chatterjee, D., et al., J. Biol Chem 266, 9652-9660 (1991)).
EMB inhibits the in vivo conversion of [.sup.14 C]glucose into cell wall arabinan (Takayama, K. & Kolburn, J. O., Antimicrob Agents Chemother 33, 143-1499 (1989)), and results in the accumulation of the lipid carrier decaprenyl-P-arabinose (Wolucka, B. A., et al., J Biol Chem 269, 23328-23335 (1994)), which suggest that the drug interferes with the transfer of arabinose to the cell wall acceptor. The synthesis of lipoarabinomannan is also inhibited in the presence of EMB (Deng, L., et al. Antimicrob Agents Chemother 39, 694-701 (1995)), (Mikusova, K., et al., Antimicrob Agents Chemother 39, 2484-2489 (1995)); again, this indicates a specific effect on arabinan biosynthesis.
Thus, there is a need for the identification and characterization of new target genes and proteins in strains of mycobacteria that exhibit resistance to drugs. This would require the identification of genes that participate in the biosynthesis of the mycobacterial cell wall and the identification of mutants of these genes encoding proteins that confer resistance to drugs. Characterization of the molecular target(s) of EMB could provide information on targets for new chemotherapeutic agents, and facilitate development of diagnostic strategies for the detection of resistant strains of mycobacteria.